产品内容
| S665679 | Component | 100 U | 500 U | Storage | | S665679A | Super Kfx DNA Polymerase, 2 U/μL | 50 μL | 250 μL | -20℃. Avoid freeze/thaw cycle. | | S665679B | 2×Super Kfx Buffer | 2×1.25 mL | 7×1.8 mL | -20℃. Avoid freeze/thaw cycle. | | S665679C | dNTP Mix, 10 mM each | 150 μL | 750 μL | -20℃. Avoid freeze/thaw cycle. |
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产品简介
Super Kfx DNA Polymerase是一种快速、高扩增效率的高保真DNA聚合酶，该聚 合酶具有5′-3′DNA聚合酶活性和3′-5′外切酶活性。该酶经其他高保真酶改造而来，添加 了独特的延伸因子、特异性促进因子，极大提高了扩增能力，克服了普通Pfu酶扩增能 力差、产量低和扩增速度慢的缺陷，缩短了反应时间。本品可用于普通片段、长片段 及其他各种复杂模板的扩增，扩增得到的PCR产物的3′端不带有“A”碱基，如需进行T/A 克隆，需在PCR产物末端添加“A”后进行克隆。本产品适用于基因克隆、二代建库扩 增、基因定点突变、SNP等扩增实验。
活性定义
在74℃，30分钟内，将10 nmoL脱氧核苷酸掺入到酸性不溶物质所需的酶量定义 为1个活性单位（U）。
质量控制
经过多次柱纯化，SDS-PAGE检测其纯度大于98%；经检测无外源核酸酶活性； 室温存放一个月，无明显活性改变。
使用方法
以下举例为常规PCR反应体系和反应条件，实际操作中应根据模板、引物结构和目的 片段大小不同进行相应的改进和优化。
1. PCR 反应体系
所有操作请在冰上进行，各组分解冻后请充分混匀，用完之后请及时放回-20℃保存。
试剂 | 50 μL反应体系 | 终浓度 | 2×Super Kfx Buffer | 25 μL | 1× | dNTP Mix，10 mM each | 1.5-2.5 μL | 300-500 μM each | Forward Primer，10 μM | 2 μL | 0.4 μM | Reverse Primer，10 μM | 2 μL | 0.4 μM | Template DNA适量 | 适量 | <500 ng/50 μL | Super Kfx DNA Polymerase | 0.5-0.75 μL | 1-1.5 U/50 μL | ddH2O | up to 50 μL | |
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2. PCR 反应条件
步骤 | 温度 | 时间 | | 预变性 | 98°C | 30 s-3 min | | 变性 退 | 98°C | 10-30 s | 25-35循环 | 火 延伸 | 根据引物Tm定 | 15-30 s | 25-35循环 | | 72°C | 4-6 kb/min | 25-35循环 | 终延伸 | 72°C | 5 min |
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注意：
1）优先使用三步法扩增，三步法无法扩增目的产物或引物Tm值大于68°C，请尝试两步法。
2）变性：简单模板的预变性98℃，30s-1min，对于复杂的模板，预变性时间可延长至3min。
3）退火：一般实验中退火温度比引物的Tm值低3-5℃，如无法得到理想的扩增效率时，应梯度改变退 火温度，进行优化；发生非特异性反应时，适当提高退火温度。
4）延伸：延伸时间应根据所扩增片段的长度和模板复杂程度设定，本产品扩增效率为4-6kb/min， 对于长片段及复杂性高的模板建议2-4kb/min。
5）循环次数：可根据扩增产物的下游应用设定循环数，如果循环次数太少，扩增量不足，循环次数太 多，错配机率会增加，所以在保证产物得率的前提下应尽量减少循环次数。
Products content
| S665679 | Component | 100 U | 500 U | Storage | | S665679A | Super Kfx DNA Polymerase, 2 U/μL | 50 μL | 250 μL | -20℃. Avoid freeze/thaw cycle. | | S665679B | 2×Super Kfx Buffer | 2×1.25 mL | 7×1.8 mL | -20℃. Avoid freeze/thaw cycle. | | S665679C | dNTP Mix, 10 mM each | 150 μL | 750 μL | -20℃. Avoid freeze/thaw cycle. |
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Products Introduction
Super Kfx DNA Polymerase is a high-fidelity DNA polymerase with 5′-3′ DNA polymerase activity and 3′-5′ exonuclease activity for fast and efficient amplification. The polymerase has 5′-3′ DNA polymerase activity and 3′-5′ exonuclease activity. The enzyme has been modified from other high-fidelity enzymes by adding unique extension factors and specific promoter factors, which greatly improves the amplification ability, overcomes the defects of ordinary Pfu enzyme's poor amplification ability, low yield, and slow amplification speed, and shortens the reaction time. This product can be used for the amplification of ordinary fragments, long fragments and other complex templates, the 3′ end of the amplified PCR product does not have the "A" base, if you need to carry out T/A cloning, you need to add the "A" at the end of the PCR product and then clone it. This product is suitable for gene cloning, second generation library amplification, targeted gene mutation, SNP amplification experiments.
Active Definition
The amount of enzyme required to incorporate 10 nmoL of deoxyribonucleotide into an acid-insoluble substance is defined as 1 active unit (U) at 74°C for 30 min.
Quality control
After several column purifications, the purity of the product was greater than 98% by SDS-PAGE; no exogenous nuclease activity was detected; and the product was stored at room temperature for one month without any obvious activity change.
Usage
The following are examples of conventional PCR reaction systems and conditions, which should be improved and optimized according to the template, primer structure and fragment size.
1. PCR reaction system
All operations should be carried out on ice, please mix the components thoroughly after thawing, after use, please promptly return to -20 ℃ for storag
2. PCR reaction system
Take note of
1）Priority is given to three-step amplification; if the three-step method fails to amplify the target product or if the primer Tm value is greater than 68°C, try the two-step method.
2）Denaturation: pre-denaturation of simple templates 98°C, 30s-1min, for complex templates, the pre-denaturation time can be extended to 3min.
3）Annealing: In general, the annealing temperature is 3-5℃ lower than the Tm value of the primers. If the desired amplification efficiency cannot be obtained, the annealing temperature should be changed in a gradient to optimize the results; if a non-specific reaction occurs, the annealing temperature should be increased appropriately.
4）Extension: The extension time should be set according to the length of the amplified fragments and the complexity of the template, the amplification efficiency of this product is 4-6kb/min, for long fragments and templates with high complexity it is recommended that 2-4kb/min.
5）Cycling times: the number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too small, the amplification amount will be insufficient, and if the number of cycles is too large, the chance of mismatch will be increased, so the number of cycles should be minimized under the premise of guaranteeing the yield of the product.
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